T cell receptor mutants

ABSTRACT

A T cell receptor (TCR) having the property of binding to EVDPIGHLY HLA-A1 complex and comprising a specified wild type TCR which has specific mutations in the TCR alpha variable domain and/or the TCR beta variable domain to increase affinity. Such TCRs are useful for adoptive therapy.

This application claims the benefit of and incorporates by referencePCT/GB2010/001433 filed on 28 Jul. 2010.

This application incorporates by reference the contents of a 23.4 kbtext file created on Aug. 4, 2010 and named “sequence_listing.txt” whichis the sequence listing for this application.

The present invention relates to T cell receptors (TCRs) which bind theEVDPIGHLY (SEQ ID NO: 1) peptide (derived from the MAGE-3 protein)presented as a peptide-HLA-A 1 complex, the TCRs being mutated relativeto the native MAGE-A3 TCR alpha and/or beta variable domains and havingbinding affinities for, and/or binding half-lives for, the complex atleast double that of a reference MAGE-A3 TCR.

BACKGROUND TO THE INVENTION

The EVDPIGHLY (SEQ ID No: 1) peptide corresponds to amino acid residuenumbers 168-176 of the known MAGE-3 protein. The MAGE-3 protein isexpressed in many tumour types, including melanomas, and other solidtumours such as Head and Neck Squamous Cell, lung, bladder, gastric andesophageal carcimomas. The MAGE-3 peptide EVDPIGHLY (SEQ ID No: 1) isthe best characterised MAGE-3 epitope. It is recognised by both HLA-A1and HLA-B35 restricted T cells. It is able to elicit cytotoxic activityagainst peptide-pulsed, HLA-A1 positive target cells, andMAGE-3-expressing HLA-A1 positive melanoma cell lines. The peptide, usedas a vaccine, has been shown to induce tumour regression and elicit CTLresponses in some of those patients.

Therefore, the EVDPIGHLY (SEQ ID NO: 1) HLA-A1 complex provides a cancermarker that the TCRs of the invention can target. For example, TCRs ofthe invention may be transformed into T-cells, rendering them capable ofdestroying tumour cells presenting that HLA complex, for administrationto a patient in the treatment process known as adoptive therapy. Forthis purpose it would be desirable if the TCRs had a higher affinityand/or a slower off-rate for the peptide-HLA complex than native TCRsspecific for that complex. Dramatic increases in affinity have beenassociated with a loss of antigen specificity in TCR gene-modified CD8 Tcells, which could result in the nonspecific activation of theseTCR-transfected CD8 T cells, so TCRs having somewhat a higher affinityand/or a slower off-rate for the peptide-HLA complex than native TCRsspecific for that complex, but not a dramatically higher affinity and/ordramatically slower off-rate for the peptide-HLA than native TCRs, wouldbe preferred for adoptive therapy (see Zhao et al., (2007) J. Immunol.179: 5845-54; Robbins et al., (2008) J. Immunol. 180: 6116-31; and seealso published WO 2008/038002).

TCRs are described using the International Immunogenetics (IMGT) TCRnomenclature, and links to the IMGT public database of TCR sequences.Native alpha-beta heterodimeric TCRs have an alpha chain and a betachain. Broadly, each chain comprises variable, joining and constantregions, and the beta chain also usually contains a short diversityregion between the variable and joining regions, but this diversityregion is often considered as part of the joining region. Each variableregion comprises three CDRs (Complementarity Determining Regions)embedded in a framework sequence, one being the hypervariable regionnamed CDR3. There are several types of alpha chain variable (Vα) regionsand several types of beta chain variable (Vβ) regions distinguished bytheir framework, CDR1 and CDR2 sequences, and by a partly defined CDR3sequence. The Vα types are referred to in IMGT nomenclature by a uniqueTRAV number. Thus “TRAV21” defines a TCR Vα region having uniqueframework and CDR1 and CDR2 sequences, and a CDR3 sequence which ispartly defined by an amino acid sequence which is preserved from TCR toTCR but which also includes an amino acid sequence which varies from TCRto TCR. In the same way, “TRBV5-1” defines a TCR Vβ region having uniqueframework and CDR1 and CDR2 sequences, but with only a partly definedCDR3 sequence.

The joining regions of the TCR are similarly defined by the unique IMGTTRAJ and TRBJ nomenclature, and the constant regions by the IMGT TRACand TRBC nomenclature.

The beta chain diversity region is referred to in IMGT nomenclature bythe abbreviation TRBD, and, as mentioned, the concatenated TRBD/TRBJregions are often considered together as the joining region.

The α and β chains of αβ TCR's are generally regarded as each having two“domains”, namely variable and constant domains. The variable domainconsists of a concatenation of variable region and joining region. Inthe present specification and claims, the term “TCR alpha variabledomain” therefore refers to the concatenation of TRAV and TRAJ regions,and the term TCR alpha constant domain refers to the extracellular TRACregion, or to a C-terminal truncated TRAC sequence. Likewise the term“TCR beta variable domain” refers to the concatenation of TRBV andTRBD/TRBJ regions, and the term TCR beta constant domain refers to theextracellular TRBC region, or to a C-terminal truncated TRBC sequence.

The unique sequences defined by the IMGT nomenclature are widely knownand accessible to those working in the TCR field. For example, they canbe found in the IMGT public database. The “T cell Receptor Factsbook”,(2001) LeFranc and LeFranc, Academic Press, ISBN 0-12-441352-8 alsodiscloses sequences defined by the IMGT nomenclature, but because of itspublication date and consequent time-lag, the information thereinsometimes needs to be confirmed by reference to the IMGT database.

We have confirmed that a native MAGE-3 TCR (Clone EB81-103 from Dr.Pierre G. Coulie, Cellular Genetics Unit, University of Louvain, AvenueHippocrate 74, UCL 7459, B-1200 Brussels, Belgium; see also Karanikas,et. al. (2003) “Monoclonal anti-MAGE-3 CTL responses in melanomapatients displaying tumor regression after vaccination with arecombinant canarypox virus.” J. Immunol. 171(9): 4898-904)) has thefollowing alpha chain and beta chain V, J and C gene usage:

-   -   Alpha chain -TRAV21*01/TRAJ28/TRAC (the extracellular sequence        of the native MAGE-A3 TCR alpha chain is given in SEQ ID No: 2)

Beta chain: -TRBV5-1*01/TRBD1/TRBJ2-7*01/TRBC2 (the extracellularsequence of the native MAGE-A3 TCR beta chain is given in SEQ ID No: 3).(Note that the TRBV5-1 sequence has 2 allelic variants, designated inIMGT nomenclature as TRBV5-1*01 and *02 respectively, and the nativeMAGE-A3 TCR clone referred to above has the * 01 variation. In the sameway, the TRBJ2-7 sequence has two known variations and it is the *01sequence which is present in the TCR clone referred to above. Note alsothat the absence of a “*” qualifier means that only one allele is knownfor the relevant sequence.)

The terms “wild type TCR”, “native TCR”, “wild type MAGE-A3 TCR” and“native MAGE-A3 TCR” are used synonymously herein to refer to thisnaturally occurring TCR having the extracellular alpha and beta chainSEQ ID Nos: 2 and 3.

DETAILED DESCRIPTION OF THE INVENTION

According to the invention, there is provided a T cell receptor (TCR)having the property of binding to EVDPIGHLY (SEQ ID No: 1) HLA-A1complex and comprising a TCR alpha variable domain and a TCR betavariable domain, characterized in that:

the TCR alpha variable domain has the amino acid sequence from K1 toP114 of SEQ ID No: 2 except that at least one of the following mutationsis present, namely

50I is mutated to 50V;

51Q is mutated to 51R;

52S is mutated to 52P;

53S is mutated to 53Y; and/or

the TCR beta variable domain has the amino acid sequence from K1 to T112of SEQ ID No: 3 except that at least one of the following mutations ispresent, namely

50F is mutated to 50T;

51S is mutated to 51D;

52E is mutated to 52M;

53T is mutated to 53L;

54Q is mutated to 54L.

TCRs of the invention preferably have a binding affinity for, and/or abinding half-life for, the EVDPIGHLY (SEQ ID NO: 1)-HLA-A1 complex atleast double that of a reference MAGE-A3 TCR, the said reference MAGE-A3TCR having the extracellular alpha chain sequence SEQ ID No: 6 and theextracellular beta chain sequence SEQ ID No: 7.

Note that SEQ ID No: 6 is the native alpha chain extracellular sequenceID No: 2 except that C162 has been substituted for T162 (i.e. T48 ofTRAC). Likewise SEQ ID No: 7 is the native beta chain extracellularsequence ID No: 3 except that C169 has been substituted for S169 (i.e.S57 of TRBC2), A187 has been substituted for C187 and D201 has beensubstituted for N201. These cysteine substitutions relative to thenative alpha and beta chain extracellular sequences enable the formationof an interchain disulfide bond which stabilises the refolded solubleTCR, ie the TCR formed by refolding extracellular alpha and beta chains.Use of the stable disulfide linked soluble TCR as the reference TCRenables more convenient assessment of binding affinity and binding halflife. The other mutations in the alpha chain SEQ ID No: 6 and the betachain SEQ ID No: 7 relative to the native alpha and beta chains SEQ IDNos: 2 and 3 are “silent” in the sense that they do not affect thebinding affinity or binding half life relative the native sequence.Hence, if a TCR of the invention has a binding affinity for, and/or abinding half-life for, the EVDPIGHLY (SEQ ID NO: 1)-HLA-A1 complex atleast double that of the reference MAGE-A3 TCR, it impliedly also meetsthose criteria with respect to the native MAGE-A3 TCR clone referred toabove.

The “reference MAGE-A3 TCR having the extracellular alpha chain sequenceSEQ ID No: 6 and the extracellular beta chain sequence SEQ ID No: 7” isreferred to synonymously hereafter either as “the reference TCR” or “thereference MAGE-A3 TCR”.

Binding affinity (inversely proportional to the equilibrium constantK_(D)) and binding half-life (expressed as T½) can be determined by anyappropriate method. It will be appreciated that doubling the affinity ofa TCR results in halving the K_(D). T½ is calculated as ln 2 divided bythe off-rate (k_(off)). So doubling of T½ results in a halving ink_(off). K_(D) and k_(off) values for TCRs are usually measured forsoluble forms of the TCR, i.e. those forms which are truncated to removehydrophobic transmembrane domain residues. Therefore it is to beunderstood that a given TCR meets the requirement that it has a bindingaffinity for, and/or a binding half-life for, the EVDPIGHLY (SEQ ID NO:1)-HLA-A1 complex if a soluble form of that TCR meets that requirement.Preferably the binding affinity or binding half-life of a given TCR ismeasured several times, for example 3 or more times, using the sameassay protocol, and an average of the results is taken. In a preferredembodiment these measurements are made using the Surface PlasmonResonance (BIAcore) method of Example 3 herein. The reference MAGE-A3TCR has a K_(D) of approximately 250 μM as measured by that method, andthe k_(off) was approximately 0.2 s⁻¹ (i.e T½ was approximately 3 s).

The TCRs of the invention have an affinity and/or a binding half-lifefor the EVDPIGHLY (SEQ ID NO: 1) HLA-A1 complex at least twice that ofthe reference MAGE-A3 TCR, while retaining acceptable EVDPIGHLY (SEQ IDNO: 1) HLA-A1 complex specificity, for example similar to the referenceMAGE A3 TCR. TCRs required for transfection of T-cells for adoptivetherapy should have somewhat higher affinities and/or longer bindinghalf-lives for the said EVDPIGHLY (SEQ ID NO: 1) HLA-A1 complex than thereference MAGE-A3 TCR (though still respectively at least twice those ofthe native TCR).

For example, TCRs of the invention may have a K_(D) for the complex offrom about 6 μM to about 70 μM and/or have a binding half-life (T½) forthe complex of from about 1 to about 11 s.

For the purposes of the present invention, a TCR is a moiety having atleast one TCR alpha and/or TCR beta variable domain. Generally theycomprise both a TCR alpha variable domain and a TCR beta variabledomain. They may be αβ heterodimers or may be single chain format. Foruse in adoptive therapy, an αβ heterodimeric TCR may, for example, betransfected as full length chains having both cytoplasmic andtransmembrane domains. If desired, an introduced disulfide bond betweenresidues of the respective constant domains may be present (see forexample WO 2006/000830).

Whatever the format, the TCRs of the invention are mutated relative tothe native MAGE-A3 TCR having the extracellular alpha and beta chainsequences SEQ ID Nos: 2 and 3 in their alpha variable domain (extendingfrom K1 to P114 of SEQ ID No: 2) and/or beta variable domain (extendingfrom K1 to T112 of SEQ ID No: 3). The native MAGE-A3 or the referenceMAGE-A3 TCR can be used as a template into which the various mutationsthat cause high affinity and/or a slow off-rate for the interactionbetween TCRs of the invention and the EVDPIGHLY (SEQ ID NO: 1) HLA-A1complex can be introduced. Embodiments of the inventions include TCRswhich are mutated relative to the a chain variable domain extending fromK1 to P114 of SEQ ID No: 2 and/or 13 chain variable domain extendingfrom K1 to T112 of SEQ ID No: 3 in at least one complementaritydetermining region (CDR) and/or variable domain framework regionthereof.

Mutations can be carried out using any appropriate method including, butnot limited to, those based on polymerase chain reaction (PCR),restriction enzyme-based cloning, or ligation independent cloning (LIC)procedures. These methods are detailed in many of the standard molecularbiology texts. For further details regarding polymerase chain reaction(PCR) mutagenesis and restriction enzyme-based cloning see Sambrook &Russell, (2001) Molecular Cloning—A Laboratory Manual (3^(rd) Ed.) CSHLPress. Further information on LIC procedures can be found in(Rashtchian, (1995) Curr Opin Biotechnol 6 (1): 30-6).

One method for generating high affinity MAGE-3 TCRs of the invention isselection from a diverse library of phage particles displaying such TCRsas disclosed in WO 2004/044004.

It should be noted that any αβ TCR that comprises similar Vα and Vβ geneusage and therefore variable domain amino acid sequences to that of thenative MAGE-A3 TCR or reference MAGE-3 TCR could make a convenienttemplate TCR. It would then be possible to introduce into the DNAencoding one or both of the variable domains of the template αβ TCR thechanges required to produce the mutated TCRs of the invention. As willbe obvious to those skilled in the art, the necessary mutations could beintroduced by a number of methods, for example site-directedmutagenesis.

In some embodiments, the TCRs of the invention have the alpha chainvariable domain extending from K1 to P114 of SEQ ID No: 2, except thatamino acid residues at one or more of positions 50I, 51Q, 52S or 53S aremutated, and/or having the beta chain variable domain extending from K1to T112 of SEQ ID No: 3, except that amino acid residues at one or moreof positions 50F, 51S, 52E, 53T or 54Q are mutated. For example, TCRs ofthe invention may have one or more of alpha chain variable domain aminoacid residues 50V, 51R, 52P or 53Y using the numbering shown in SEQ IDNo: 2, and/or one or more of beta chain variable domain amino acidresidues 50T, 51D, 52M, 53L, or 54L using the numbering shown in SEQ IDNo: 3.

Specific TCRs of the invention include those comprising one of the alphachain variable domain amino acid sequences SEQ ID Nos: 8 and 9 and/orone of the beta chain variable domain amino acid sequences SEQ ID Nos:10 and 11. Thus TCRs with the variable domain sequence of the wild typealpha chain (K1 to P114 of SEQ ID No: 2) may be associated with a betachain having one of SEQ ID Nos: 10 and 11. Alternatively, an alpha chainhaving one of SEQ ID Nos: 8 and 9 may be associated with the variabledomain sequence of the wild type beta chain (K1 to T112 of SEQ ID No:3). Alternatively an alpha chain having one of SEQ ID Nos: 8 and 9 maybe associated with a beta chain having one of SEQ ID Nos: 10 and 11.

Phenotypically silent variants of the TCRs discussed above also formpart of this invention. The term “phenotypically silent variants” refersto TCRs which are identical in sequence to a TCR of the invention exceptthat they incorporate changes in the constant and/or variable domainswhich do not alter the affinity and/or off-rate for the interaction withthe peptide-HLA complex. One example of such a variant is provided byTCRs of the invention in which the TCR alpha constant domain contains aPhenylalanine (F) amino acid residue substituted for the 135 Serine (S)amino acid residue using the numbering of SEQ ID No: 2.

As mentioned above, αβ heterodimeric TCRs of the invention may have anintroduced disulfide bond between their constant domains. Preferred TCRsof this type include those which have a TRAC constant domain sequenceand a TRBC1 or TRBC2 constant domain sequence except that Thr 48 of TRACand Ser 57 of TRBC1 or TRBC2 are replaced by cysteine residues, the saidcysteines forming a disulfide bond between the TRAC constant domainsequence and the TRBC1 or TRBC2 constant domain sequence of the TCR.

With or without the introduced inter-chain bond mentioned in thepreceding paragraph, αβ heterodimeric TCRs of the invention may have aTRAC constant domain sequence and a TRBC1 or TRBC2 constant domainsequence, and the TRAC constant domain sequence and the TRBC1 or TRBC2constant domain sequence of the TCR may be linked by the nativedisulfide bond between Cys4 of exon 2 of TRAC and Cys2 of exon 2 ofTRBC1 or TRBC2.

Since the αβ heterodimeric TCRs of the invention have utility inadoptive therapy, the invention includes an isolated cell, especially aT-cell, presenting a TCR of the invention. There are a number of methodssuitable for the transfection of T cells with DNA or RNA encoding theTCRs of the invention. (See for example Robbins et al., (2008) J.Immunol. 180: 6116-6131)). T cells expressing the TCRs of the inventionwill be suitable for use in adoptive therapy-based treatment of MAGE-3⁺HLA-A1⁺ cancers. As will be known to those skilled in the art there area number of suitable methods by which adoptive therapy can be carriedout. (See for example Rosenberg et al., (2008) Nat Rev Cancer 8 (4):299-308).

For use in adoptive therapy, the invention also includes cellsharbouring a TCR expression vector which comprises nucleic acid encodingthe TCR of the invention in a single open reading frame or two distinctopen reading frames. Also included in the scope of the invention arecells harbouring a first expression vector which comprises nucleic acidencoding the alpha chain of a TCR of the invention, and a secondexpression vector which comprises nucleic acid encoding the beta chainof a TCR of the invention,

The TCRs of the invention intended for use in adoptive therapy areglycosylated when expressed by the transfected T cells. As is wellknown, the glycosylation pattern of transfected TCRs may be modified bymutations of the transfected gene.

For administration to patients, T cells transfected with TCRs of theinvention may be provided in pharmaceutical composition together with apharmaceutically acceptable carrier. Cells in accordance with theinvention will usually be supplied as part of a sterile, pharmaceuticalcomposition which will normally include a pharmaceutically acceptablecarrier. This pharmaceutical composition may be in any suitable form,(depending upon the desired method of administering it to a patient). Itmay be provided in unit dosage form, will generally be provided in asealed container and may be provided as part of a kit. Such a kit wouldnormally (although not necessarily) include instructions for use. It mayinclude a plurality of said unit dosage forms.

The pharmaceutical composition may be adapted for administration by theintravenous route. Such compositions may be prepared by any method knownin the art of pharmacy, for example by mixing the active ingredient withthe carrier(s) or excipient(s) under sterile conditions.

Dosages of the substances of the present invention can vary between widelimits, depending upon the disease or disorder to be treated, the ageand condition of the individual to be treated, etc. and a physician willultimately determine appropriate dosages to be used.

EXAMPLES

The invention is further described in the following examples in whichthe following Figures are referred to.

FIGS. 1A and 2A respectively show the extracellular amino acid sequencesof the native MAGE-A3 TCR alpha chain having the TRAV21*01/TRAJ28/TRACgene usage, and of the native MAGE-A3 TCR beta chain having theTRBV5-1*01/TRBD1/TRBJ2-7*01/TRBC2 gene usage (SEQ ID Nos: 2 and 3respectively).

FIGS. 1B and 2B respectively show DNA sequences encoding solublewild-type MAGE-A3 TCR alpha and beta chains also referred to as thereference MAGE-A3 TCR alpha and beta chains. These sequences includeadditional cysteine residues to form a non-native disulphide bond. Themutated codons encoding the additional cysteine residues are bold. TheNdeI and HindIII restriction enzyme recognition sequences areunderlined.

FIGS. 1C and 2C respectively show the soluble wild-type MAGE-A3 TCR, orreference MAGE-A3 TCR, alpha and beta chain extracellular amino acidsequences (SEQ ID Nos: 6 and 7 respectively) produced from the DNAsequences of FIGS. 1B and 2B respectively, but without the introducedleading methionine inserted for efficient expression in bacteria. Theintroduced cysteines are bold and underlined.

FIGS. 3A-B show the alpha chain variable domain amino acid sequences ofMAGE-A3 TCR variants in accordance with the invention. The mutatedresidues are bold and underlined.

FIGS. 4A-B show the beta chain variable domain amino acid sequences ofMAGE-A3 TCR variants in accordance with the invention. The mutatedresidues are bold and underlined.

FIG. 5A shows the DNA sequence for the wild type MAGE-A3-specific TCRgene (WT alpha chain-2A-WT beta chain construct with the Porcineteschovirus-1 2A sequence bold and underlined) for transduction ofT-cells.

FIG. 5B shows the amino acid sequence of the wild type MAGE-A3-specificTCR for T-cell transduction produced from the DNA sequence of FIG. 5.The Porcine teschovirus-1 2A sequence is bold and underlined.

FIG. 6 shows the IFN-γ release of MAGE-A3 TCR-transduced T-cells inresponse to a range of target cells in an ELISPOT assay. These figuresshow the increased specific activation of T cells transduced with higheraffinity MAGE-A3 TCRs compared to T cells transduced with the nativeMAGE-A3 TCR.

FIG. 7 shows a cytotoxicity assay where killing of tumour cell lines byMAGE-A3-transduced T-cells is tested.

EXAMPLE 1 Cloning of the Reference MAGE-A3 TCR Alpha and Beta ChainVariable Region Sequences into pGMT7-Based Expression Plasmids

The reference MAGE-A3 TCR variable alpha and TCR variable beta domainswere PCR amplified from total cDNA isolated from a MAGE-3 T cell clone(Clone EB81-103 from Pierre Coulie University of Louvain, Belgium). Inthe case of the alpha chain, an alpha chain variable region sequencespecific oligonucleotide A1 (ggaattccatatgaaacaagaagttactcaaattcc SEQ IDNo: 14) which encodes the restriction site NdeI and an introducedmethionine for efficient initiation of expression in bacteria, and analpha chain constant region sequence specific oligonucleotide A2(ttgtcagtcgacttagagtctctcagctggtacacg SEQ ID No: 15) which encodes therestriction site SalI are used to amplify the alpha chain variableregion. In the case of the beta chain, a beta chain variable regionsequence specific oligonucleotide B1(gaattccatatgaaagctggagttactcaaactccaag SEQ ID No: 16) which encodes therestriction site NdeI and an introduced methionine for efficientinitiation of expression in bacteria, and a beta chain constant regionsequence specific oligonucleotide B2 (tagaaaccggtggccaggcacaccagtgtggcSEQ ID No: 17) which encodes the restriction site AgeI are used toamplify the beta chain variable region.

The alpha and beta variable regions were cloned into pGMT7-basedexpression plasmids containing either Cα or Cβ by standard methodsdescribed in Molecular Cloning a Laboratory Manual Third edition bySambrook and Russell. Plasmids were sequenced using an AppliedBiosystems 3730×1 DNA Analyzer.

The DNA sequences encoding the TCR alpha chain cut with NdeI and SalIwere ligated into pGMT7+Cα vector, which was cut with NdeI and XhoI. TheDNA sequences encoding the TCR beta chain cut with NdeI and AgeI wasligated into separate pGMT7+Cβ vector, which was also cut with NdeI andAgeI.

Ligation

Ligated plasmids were transformed into competent Escherichia coli strainXL1-blue cells and plated out on LB/agar plates containing 100 μg/mlampicillin. Following incubation overnight at 37° C., single coloniesare picked and grown in 10 ml LB containing 100 μg/ml ampicillinovernight at 37° C. with shaking. Cloned plasmids were purified using aMiniprep kit (Qiagen) and the plasmids were sequenced using an AppliedBiosystems 3730×1 DNA Analyzer.

FIGS. 1C and 2C show respectively the soluble disulfide-linked referenceMAGE-A3 TCR α and β chain extracellular amino acid sequences (SEQ IDNos: 6 and 7 respectively) produced from the DNA sequences of FIGS. 1Band 2B respectively, but without the introduced leading methionineinserted for efficient expression in bacteria. Note that cysteines weresubstituted in the constant regions of the alpha and beta chains toprovide an artificial inter-chain disulphide bond on refolding to formthe heterodimeric TCR. The introduced cysteines are shown in bold andunderlined. The restriction enzyme recognition sequences in the DNAsequences of FIGS. 1B and 2B are underlined.

EXAMPLE 2 Expression, Refolding and Purification of Soluble ReferenceMAGE-A3 TCR

The expression plasmids containing the TCR α-chain and β-chainrespectively, as prepared in Example 1, were transformed separately intoE. coli strain Rosetta (DE3)pLysS, and single ampicillin-resistantcolonies were grown at 37° C. in TYP (ampicillin 100 μg/ml) medium toOD₆₀₀ of ˜0.6-0.8 before inducing protein expression with 0.5 mM IPTG.Cells were harvested three hours post-induction by centrifugation for 30minutes at 4000 rpm in a Beckman J-6B. Cell pellets were lysed with 25ml Bug Buster (NovaGen) in the presence of MgCl₂ and DNaseI. Inclusionbody pellets were recovered by centrifugation for 30 minutes at 13000rpm in a Beckman J2-21 centrifuge. Three detergent washes were thencarried out to remove cell debris and membrane components. Each time theinclusion body pellet was homogenised in a Triton buffer (50 mM Tris-HClpH 8.0, 0.5% Triton-X100, 200 mM NaCl, 10 mM NaEDTA) before beingpelleted by centrifugation for 15 minutes at 4,000 rpm. Detergent andsalt was then removed by a similar wash in the following buffer: 50 mMTris-HCl pH 8.0, 1 mM NaEDTA, pH8.0. Finally, the inclusion bodies weredivided into 30 mg aliquots and frozen at −70° C. Inclusion body proteinyield was quantified by solubilising with 6 M Guanidine-HCl and an ODmeasurement was taken on a Hitachi U-2001 Spectrophotometer. The proteinconcentration was then calculated using the extinction coefficient.

Approximately 15 mg of TCR β chain and 15 mg of TCR α chain solubilisedinclusion bodies were thawed from frozen stocks and diluted into 10 mlof a guanidine solution (6 M Guanidine-hydrochloride, 50 mM Tris HCl pH8.1, 100 mM NaCl, 10 mM EDTA, 10 mM DTT), to ensure complete chaindenaturation. The guanidine solution containing fully reduced anddenatured TCR chains was then injected into 0.5 litre of the followingrefolding buffer: 100 mM Tris pH 8.1, 400 mM L-Arginine, 2 mM EDTA, 5 MUrea. The redox couple (cysteamine hydrochloride and cystaminedihydrochloride) to final concentrations of 6.6 mM and 3.7 mMrespectively, were added approximately 5 minutes before addition of thedenatured TCR chains. The solution was left for ˜30 minutes. Therefolded TCR was dialysed in dialysis tubing cellulose membrane(Sigma-Aldrich; Product No. D9402) against 10 L H₂O at 5° C.±3° C. for18-20 hours. After this time, the dialysis buffer was changed twice tofresh 10 mM Tris pH 8.1 (10 L) and dialysis was continued at 5° C.+3° C.for another ˜8 hours.

Soluble TCR was separated from misfolded, degradation products andimpurities by loading the dialysed refold onto a POROS 50HQ anionexchange column and eluting bound protein with a gradient of 0-500 mMNaCl in 10 mM Tris pH 8.1 over 6 column volumes using an Akta purifier(GE Healthcare). Peak fractions were then stored at 4° C. and analysedby Coomassie-stained SDS-PAGE before being pooled and concentrated.Finally, the soluble TCR was purified and characterised using a GEHealthcare Superdex 75HR gel filtration column pre-equilibrated in PBSbuffer (Sigma). The peak eluting at a relative molecular weight ofapproximately 50 kDa was pooled and concentrated prior tocharacterisation by BIAcore surface plasmon resonance analysis.

EXAMPLE 3 Binding Characterisation

BIAcore Analysis

A surface plasmon resonance biosensor (BIAcore 3000™) can be used toanalyse the binding of a soluble TCR to its peptide-MHC ligand. This isfacilitated by producing soluble biotinylated peptide-HLA (“pHLA”)complexes which can be immobilised to a streptavidin-coated bindingsurface (sensor chip). The sensor chips comprise four individual flowcells which enable simultaneous measurement of T-cell receptor bindingto four different pHLA complexes. Manual injection of pHLA complexallows the precise level of immobilised class I molecules to bemanipulated easily.

Biotinylated class I HLA-A*01 molecules were refolded in vitro frombacterially-expressed inclusion bodies containing the constituentsubunit proteins and synthetic peptide, followed by purification and invitro enzymatic biotinylation (O'Callaghan et al. (1999) Anal. Biochem.266: 9-15). HLA-A*01-heavy chain was expressed with a C-terminalbiotinylation tag which replaces the transmembrane and cytoplasmicdomains of the protein in an appropriate construct. Inclusion bodyexpression levels of ˜75 mg/litre bacterial culture were obtained. TheMHC light-chain or β2-microglobulin (β2m) was also expressed asinclusion bodies in E. coli from an appropriate construct, at a level of˜500 mg/litre bacterial culture.

E. coli cells were lysed and inclusion bodies were purified toapproximately 80% purity. Synthetic peptide (MAGE-A3 EVDPIGHLY (SEQ IDNO: 1)) was dissolved in DMSO to a final concentration of 4 mg/ml.Inclusion bodies of β2m and heavy chain were denatured separately in 6 Mguanidine-HCl, 50 mM Tris pH 8.1, 100 mM NaCl, 10 mM DTT, 10 mM EDTA.Refolding buffer was prepared containing 0.4 M L-Arginine, 100 mM TrispH 8.1, 3.7 mM cystamine dihydrochloride, 6.6 mM cysteaminehydrochloride and chilled to <5° C. Preferably the peptide was addedfirst to the refold buffer, followed by addition of denatured β2m thenaddition of denatured heavy chain. The MAGE-A3 EVDPIGHLY (SEQ ID NO: 1)peptide was added to the refold buffer at 4 mg/litre (finalconcentration). Then 30 mg/litre β2m followed by 30 mg/litre heavy chain(final concentrations) were added. Refolding was allowed to reachcompletion at 4° C. for at least 1 hour.

Buffer was exchanged by dialysis in 10 volumes of 10 mM Tris pH 8.1. Twochanges of buffer were necessary to reduce the ionic strength of thesolution sufficiently. The protein solution was then filtered through a1.5 μm cellulose acetate filter and loaded onto a POROS 50HQ anionexchange column (8 ml bed volume). Protein was eluted with a linear0-500 mM NaCl gradient in 10 mM Tris pH 8.1 using an Akta purifier (GEHealthcare). HLA-A*01-peptide complex eluted at approximately 250 mMNaCl, and peak fractions were collected, a cocktail of proteaseinhibitors (Calbiochem) was added and the fractions were chilled on ice.

Biotinylation tagged pHLA molecules were buffer exchanged into 10 mMTris pH 8.1, 5 mM NaCl using a GE Healthcare fast desalting columnequilibrated in the same buffer. Immediately upon elution, theprotein-containing fractions were chilled on ice and protease inhibitorcocktail (Calbiochem) was added. Biotinylation reagents were then added:1 mM biotin, 5 mM ATP (buffered to pH 8), 7.5 mM MgCl₂, and 5 μg/ml BirAenzyme (purified according to O'Callaghan et al. (1999) Anal. Biochem.266: 9-15). The mixture was then allowed to incubate at room temperatureovernight.

The biotinylated pHLA-A*01 molecules were purified using gel filtrationchromatography. A GE Healthcare Superdex 75 HR 10/30 column waspre-equilibrated with filtered PBS and 1 ml of the biotinylationreaction mixture was loaded and the column was developed with PBS at 0.5ml/min using an Akta purifier (GE Healthcare). Biotinylated pHLA-A*01molecules eluted as a single peak at approximately 15 ml. Fractionscontaining protein were pooled, chilled on ice, and protease inhibitorcocktail was added. Protein concentration was determined using aCoomassie-binding assay (PerBio) and aliquots of biotinylated pHLA-A*01molecules were stored frozen at −20° C.

Such immobilised complexes are capable of binding both T-cell receptorsand the coreceptor CD8αα, both of which may be injected in the solublephase. The pHLA binding properties of soluble TCRs are observed to bequalitatively and quantitatively similar if the TCR is used either inthe soluble or immobilised phase. This is an important control forpartial activity of soluble species and also suggests that biotinylatedpHLA complexes are biologically as active as non-biotinylated complexes.

The BIAcore 3000™ surface plasmon resonance (SPR) biosensor measureschanges in refractive index expressed in response units (RU) near asensor surface within a small flow cell, a principle that can be used todetect receptor ligand interactions and to analyse their affinity andkinetic parameters. The BIAcore experiments were performed at atemperature of 25° C., using PBS buffer (Sigma, pH 7.1-7.5) as therunning buffer and in preparing dilutions of protein samples.Streptavidin was immobilised to the flow cells by standard aminecoupling methods. The pHLA complexes were immobilised via the biotintag. The assay was then performed by passing soluble TCR over thesurfaces of the different flow cells at a constant flow rate, measuringthe SPR response in doing so.

Equilibrium Binding Constant

The above BIAcore analysis methods were used to determine equilibriumbinding constants. Serial dilutions of the disulfide linked solubleheterodimeric form of the reference MAGE-A3 TCR were prepared andinjected at constant flow rate of 5 μl min⁻¹ over two different flowcells; one coated with ˜1000 RU of specific EVDPIGHLY (SEQ ID NO: 1)HLA-A*01 complex, the second coated with ˜1000 RU of non-specificHLA-A2-peptide (KIFGSLAFL (SEQ ID No: 18)) complex. Response wasnormalised for each concentration using the measurement from the controlcell. Normalised data response was plotted versus concentration of TCRsample and fitted to a non-linear curve fitting model in order tocalculate the equilibrium binding constant, K_(D). (Price & Dwek,Principles and Problems in Physical Chemistry for Biochemists (2^(nd)Edition) 1979, Clarendon Press, Oxford). The disulfide linked solubleform of the reference MAGE-A3 TCR (Example 2) demonstrated a K_(D) ofapproximately 250 μM. From the same BIAcore data the T½ wasapproximately 3 s.

Kinetic Parameters

The above BIAcore analysis methods were also used to determineequilibrium binding constants and off-rates.

For TCRs (see Example 4 below) K_(D) was determined by experimentallymeasuring the dissociation rate constant, k_(off), and the associationrate constant, k_(on). The equilibrium constant K_(D) was calculated ask_(off)/k_(on).

TCR was injected over two different cells one coated with ˜300 RU ofspecific EVDPIGHLY (SEQ ID NO: 1) HLA-A*01 complex, the second coatedwith ˜300 RU of non-specific HLA-A1-peptide complex. Flow rate was setat 50 μl/min. Typically 250 μl of TCR at a concentration equivalent to˜10 times the K_(D) was injected. Buffer was then flowed over until theresponse had returned to baseline or >2 hours had elapsed. Kineticparameters were calculated using BIAevaluation software. Thedissociation phase was fitted to a single exponential decay equationenabling calculation of half-life.

EXAMPLE 4 Generation of Variants of the Reference MAGE-A3 TCR

Phage display is one means by which libraries of MAGE-A3 TCR variantscan be generated in order to identify higher affinity mutants. The TCRphage display and screening methods described in (Li et al, (2005)Nature Biotech 23 (3): 349-354) were applied to the MAGE-A3 TCR ofExample 2.

TCRs with affinities and/or binding half-lives at least twice that ofthe reference MAGE-A3 TCR (and therefore impliedly at least twice thatof the native TCR) were identified, having one or more of alpha chainvariable domain amino acid residues 50V, 51R, 52P or 53Y using thenumbering shown in SEQ ID No: 2 and/or one or more of beta chainvariable domain amino acid residues 50T, 51D, 52M, 53L, or 54L using thenumbering shown in SEQ ID No: 3.

Specific examples of the amino acid sequences of the variable regions ofthe alpha chains (SEQ ID Nos: 8 and 9) and beta chains (SEQ ID Nos: 10and 11) of higher affinity TCRs are shown in FIGS. 3A-B and 4A-Brespectively. These alpha chains are mutated in CDR2 and beta chains aremutated in CDR2.

TCR heterodimers were refolded using the method of Example 2 above(including the introduced cysteines in the constant regions to providethe artificial inter-chain disulphide bond). In that way TCRs wereprepared, consisting of (a) the reference TCR beta chain, together withalpha chains which include the variable domains SEQ ID Nos: 8 and 9; (b)the reference TCR alpha chain, together with beta chains which includethe beta chain variable domains SEQ ID Nos: 10 and 11; and (c) variouscombinations of beta and alpha chains including the mutant variabledomains.

The interaction between these soluble disulfide-linked MAGE-A3 TCRs andthe EVDPIGHLY (SEQ ID NO: 1) HLA-A*01 complex was analysed using theBIAcore method described above, and the binding data is shown in Table1.

TABLE 1 TCR variable domain SEQ ID α β k_(off) (s⁻¹) T½ k_(on) (M⁻¹s⁻¹)K_(D) 8 3 0.114 6.1 sec NM 6.55 μM 9 3 0.95  <1 sec 1.7e4   55 μM 2 100.0666 10.4 sec  NM 9.43 μM 2 11 0.094 7.4 sec 1.4e4  6.7 μM

EXAMPLE 5 Transfection of T-Cells with Variants of the Native MAGE-A3TCR

(a) Lentiviral Vector Preparation by Express-in-Mediated TransientTransfection of 293T Cells

A 3_(rd) generation lentiviral packaging system is used to packagelentiviral vectors containing the gene encoding the desired TCR. 293Tcells are transfected with 4 plasmids (one lentiviral vector containingthe TCR alpha chain-P2A-TCR beta chain single ORF gene described inExample 5c, and 3 plasmids containing the other components necessary toconstruct infective but non-replicative lentiviral particles) usingExpress-In-mediated transfection (Open Biosystems).

For transfection take one T150 flask of 293T cells in exponential growthphase, with cells evenly distributed on the plate, and slightly morethan 50% confluent. Bring Express-In aliquots to room temperature. Place3 ml Serum-Free Medium (RPMI 1640+10 mM HEPES) in a sterile 15 mlconical tube. Add 174 μl of Express-In Reagent directly into theSerum-Free Medium (this provides for a 3.6:1 weight ratio of Reagent toDNA). Mix thoroughly by inverting tubes 3-4 times and incubate at roomtemperature for 5-20 minutes.

In a separate 1.5 ml microtube, add 15 μg plasmid DNA to premixedpackaging mix aliquots (containing 18 μg pRSV.REV (Rev expressionplasmid), 18 μg pMDLg/p.RRE (Gag/Pol expression plasmid), 7 μg pVSV-G(VSV glycoprotein expression plasmid), usually ˜22 and pipette up anddown to ensure homogeneity of the DNA mix. Add ˜1 ml ofExpress-In/Serum-Free Medium to the DNA mix drop wise then pipette upand down gently before transferring back to the remainder of theExpress-In/Serum-Free Medium. Invert tube 3-4 times and incubate at roomtemperature for 15-30 minutes.

Remove old culture medium from flask of cells. Add Express-In/medium/DNA(3 ml) complex to flask direct into the bottom of an upright flask of293T cells. Slowly place flask flat to cover cells and very gently rockthe flask to ensure even distribution. After 1 minute add 22 ml freshculture medium (R₁₀+HEPES: RPMI 1640, 10% heat-inactivated FBS, 1%Pen/Strep/L-glutamine, 10 mM HEPES) and carefully return to incubator.Incubate overnight at 37° C./5% CO₂. After 24 hours, proceed to harvestthe medium containing packaged lentiviral vectors.

To harvest the packaged lentiviral vectors, filter the cell culturesupernatent through a 0.45 micron nylon syringe filter, centrifuge theculture medium at 10,000 g for 18 hours (or 112,000 g for 2 hours),remove most of the supernatant (taking care not to disturb the pellet)and resuspend the pellet in the remaining few ml of supernatant (usuallyabout 2 ml from a 31 ml starting volume per tube). Snap freeze on dryice in 1 ml aliquots and store at −80° C.

(b) Transduction of T Cells with Packaged Lentiviral Vectors ContainingGene of Interest

Prior to transduction with the packaged lentiviral vectors, human Tcells (CD8 or CD4 or both depending on requirements) are isolated fromthe blood of healthy volunteers. These cells are counted and incubatedovernight in R₁₀ containing 50 U/ml IL-2 at 1×10⁶ cells per ml (0.5ml/well) in 48 well plates with pre-washed anti-CD3/CD28 antibody-coatedmicrobeads (Dynal T cell expander, Invitrogen) at a ratio of 3 beads percell.

After overnight stimulation, 0.5 ml of neat packaged lentiviral vectoris added to the desired cells. Incubate at 37° C./5% CO₂ for 3 days. 3days post-transduction count cells and dilute to 0.5×10⁶ cells/ml. Addfresh medium containing IL-2 as required. Remove beads 5-7 dayspost-transduction. Count cells and replace or add fresh mediumcontaining IL-2 at 2 day intervals. Keep cells between 0.5×10⁶ and 1×10⁶cells/ml. Cells can be analysed by flow cytometry from day 3 and usedfor functional assays (e.g. ELISpot for IFNγ release) from day 5. Fromday 10, or when cells are slowing division and reduced in size, freezecells in aliquots of at least 4×10⁶ cells/vial (at 1×10⁷ cells/ml in 90%FBS/10% DMSO) for storage.

(c) Wild Type (Wt) TCR Gene for T-Cell Transfection by Methods (a) and(b) Above

FIG. 5A is a DNA sequence (SEQ ID No: 12) encoding the native MAGE-A3TCR (codon-optimised for maximal human cell expression). It is a fulllength alpha chain (TRAV21)-Porcine teschovirus-12A-full length betachain (TRBV5-1) single open reading frame construct. The 2A sequence isunderlined, and is preceded by nucleotides encoding a furin cleavagesite to assist proteolytic removal of the 2A sequence (discussed furtherbelow in relation to FIG. 5B (SEQ ID No: 13). Peptide bond skippingduring protein translation of the mRNA at the 3′ end of the 2A sequenceproduces two proteins: 1) alpha TCR chain-2A fusion, 2) beta TCR chain.SEQ ID No: 12 includes NheI and SalI restriction sites (underlined).

FIG. 5B is the amino acid sequence (SEQ ID No: 13) corresponding to FIG.5A

In FIG. 5B:

-   -   M1-S19 is a leader sequence which is removed on maturation of        the wild type alpha chain TCR;    -   K20-S227 corresponds to the wild type alpha chain sequence SEQ        ID No: 2;    -   K20-R254 corresponds to the wild type alpha chain extracellular        domain;    -   I255-L271 is the alpha chain transmembrane region of the mature        TCR;    -   W272-S274 is the alpha chain intracellular region of the mature        TCR;    -   R277-R280 is the furin cleavage site to assist proteolytic        removal, in the Golgi apparatus, of the P2A sequence A285-P303;    -   G275, S276, S281 to G284, R304 are flexible linkers allowing        full function of the furin cleavage and P2A sequences;    -   M305-V323 is a leader sequence which is removed on maturation of        the wild type beta chain TCR;    -   K324-D565 corresponds to the wild type beta chain sequence SEQ        ID No: 3;    -   K324-E585 corresponds to the wild type beta chain extracellular        domain;    -   I586-V607 is the beta chain transmembrane region of the mature        TCR;    -   K608-G614 is the beta chain intracellular region of the mature        TCR.

(d) T-Cells Transfected with Wild Type and High Affinity MAGE TCRs

Following the procedures described in (a) and (b) above, the MAGE-A3alpha wt_(—)2A_beta wt TCR gene (SEQ ID No: 12 (FIG. 5A)) was insertedinto the pELNSxv lenti vector using the NheI and SalI restriction sitesunique to both DNA constructs, and transfected T-cells created.

Similarly, T-cells may be created by transfection with genes identicalto SEQ ID No: 12 (FIG. 5A) except that they encode (a) TCRs with thevariable domain sequence (K1 to P114) of the wild type alpha chain SEQID No: 2, associated with a beta chain variable domain having one of SEQID Nos: 10 or 11; or (b) an alpha chain variable domain having one ofSEQ ID Nos: 8 or 9 associated with the variable domain sequence (K1 toT112) of the wild type beta chain SEQ ID No: 3; or (c) an alpha chainvariable domain having one of SEQ ID Nos: 8 or 9 associated with a betachain variable domain having one of SEQ ID Nos: 10 or 11.

EXAMPLE 6 Increased Activation of MAGE-A3 Improved-AffinityTCR-Transduced T Cells Compared to Wild Type-Affinity in Response toTumour Cell Lines

Elispot Protocol

The following assay was carried out to demonstrate the activation ofTCR-transduced cytotoxic T lymphocytes (CTLs) in response to tumour celllines. IFN-γ production, as measured using the ELISPOT assay, was usedas a read-out for cytotoxic T lymphocyte (CTL) activation.

Reagents

Assay media: 10% FCS (Gibco, Cat#2011-09), 88% RPMI 1640 (Gibco,Cat#42401), 1% glutamine (Gibco Cat#25030) and 1%penicillin/streptomycin (Gibco Cat#15070-063).

Wash buffer: 0.01M PBS/0.05% Tween 20

PBS (Gibco Cat#10010)

The Human IFNγ ELISPOT PVDF-Enzymatic kit (Diaclone, France;Cat#856.051.020) contains all other reagents required. (Capture anddetection antibodies, skimmed milk powder, BSA, streptavidin-alkalinephosphatase and BCIP/NBT solution as well as the Human IFN-γ PVDFELISPOT 96 well plates)

Method

Target Cell Preparation

The target cells used in this method were natural epitope-presentingcells: HCT-116 colorectal carcinoma and NCI-H1975 non-small cell lungcarcinoma which are both HLA-A1⁺ MAGE⁺. N3 normal human epidermalmelanocytes, which are HLA-A1⁺ MAGE⁻ were used as a negative control.Sufficient target cells (50,000 cells/well) were washed bycentrifugation three times at 1200 rpm, 10 min in a Megafuge 1.0(Heraeus). Cells were then re-suspended in assay media at 10⁶ cells/ml.

Effector Cell Preparation

The effector cells (T cells) used in this method were a 1:1 mix of CD4+and CD8+ T cells (obtained by negative selection (using the CD4 and CD8Negative Isolation Kits, Dynal) from PBL). Cells were stimulated withantiCD3/CD28 coated beads (T cell expander, Invitrogen), transduced withlentiviruses carrying the gene encoding the full αβ TCR of interest(based on the construct described in Example 5 and shown in FIG. 5B) andexpanded in assay media containing 50 U/ml IL-2 until between 10 and 13days post transduction. These cells were then placed in assay mediaprior to washing by centrifugation at 1200 rpm, 10 min in a Megafuge 1.0(Heraeus). Cells were then re-suspended in assay media at a 4× the finalrequired concentration.

ELISPOTs

Plates were prepared as follows: 100 μl anti-IFN-γ capture antibody wasdiluted in 10 ml sterile PBS per plate. 100 μl of the diluted captureantibody was then aliquoted into each well. The plates were thenincubated overnight at 4° C. Following incubation the plates were washed(programme 1, plate type 2, Ultrawash Plus 96-well plate washer; Dynex)to remove the capture antibody. Plates were then blocked by adding 100μl 2% skimmed milk in sterile PBS to each well and incubating the platesat room temperature for two hours. The skimmed milk was then washed fromthe plates (programme 1, plate type 2, Ultrawash Plus 96-well platewasher, Dynex) and any remaining wash buffer was removed by flicking andtapping the ELISPOT plates on a paper towel.

The constituents of the assay were then added to the ELISPOT plate inthe following order:

50 μl of target cells 10⁶ cells/ml (giving a total of 50,000 targetcells/well).

Media sufficient to give 200 ul per well final volume (assay media).

50 μl effector cells (5,000 mixed transduced CD4/8⁺ cells/well).

The plates were then incubated overnight (37° C./5% CO₂). The next daythe plates were washed three times (programme 1, plate type 2, UltrawashPlus 96-well plate washer, Dynex) with wash buffer and tapped on papertowel to remove excess wash buffer. 100 μl primary detection antibodywas then added to each well. The primary detection antibody was preparedby adding 550 μl of distilled water to a vial of detection antibodysupplied with the Diaclone kit. 100 μl of this solution was then dilutedin 10 ml PBS/1% BSA (the volume required for a single plate). Plateswere then incubated at room temperature for at least 2 hr prior to beingwashed three times (programme 1, plate type 2, Ultrawash Plus 96-wellplate washer, Dynex) with wash buffer, excess wash buffer was removed bytapping the plate on a paper towel.

Secondary detection was performed by adding 100 μl of dilutedstreptavidin-Alkaline phosphatase to each well and incubating the plateat room temperature for 1 hour. The streptavidin-Alkaline phosphatasewas prepared by addition of 10 μl streptavidin-Alkaline phosphatase to10 ml PBS/1% BSA (the volume required for a single plate). The plateswere then washed three times (programme 1, plate type 2, Ultrawash Plus96-well plate washer, Dynex) with wash buffer and tapped on paper towelto remove excess wash buffer. 100 μl of BCIP/NBT solution, as suppliedwith the Diaclone kit, was then added to each well. During developmentplates were covered in foil and left for 5-15 min. Developing plateswere regularly checked for spots during this period to determine optimaltime to terminate the reaction. The plates were washed in a sink full oftap water to terminate the development reaction, and shaken dry prior totheir disassembly into their three constituent parts. The plates werethen dried at 50° C. for 1 hr prior to counting the spots that haveformed on the membrane using an Immunospot Plate reader (CTL; CellularTechnology Limited).

Results

IFNγ release by activated TCR-transduced T cells in response to avariety of MAGE-A3-positive and control tumour cell lines was tested byELISPOT assay (as described above). The number of ELISPOT spots observedin each well was plotted using Prism (Graph Pad).

CD4⁺, CD8⁺ or mixed CD4⁺/CD8 T cells expressing a) TCR No:1, b) TCRNo:2, c) TCR No:3, d) TCR No:4 or e) TCR No:5 (as described in the tablebelow) were incubated with MAGE-A3⁺ HLA:A1⁺ tumour cell lines HCT-116 orNCI-H1975 or with MAGE-A3⁻ HLA:A1⁺ N3 melanocytes. Non-transduced Tcells (Nt) were also used as a negative control.

TCR α variable domain TCR β variable domain TCR No SEQ ID NO: SEQ ID NO:1 K1 to P114 of SEQ ID No: 2 K1 to T112 of SEQ ID No: 3 2 9 K1 to T112of SEQ ID No: 3 3 8 K1 to T112 of SEQ ID No: 3 4 K1 to P114 of SEQ IDNo: 2 10 5 K1 to P114 of SEQ ID No: 2 11

FIG. 6 demonstrates that TCR No:1-transduced T cells did not releaseIFNγ in response to tumour cell lines. TCR No: 2-transduced T cellsreleased IFNγ in response to HCT-116 colorectal carcinoma cells only.

Improved-affinity MAGE-A3 TCR No: 3-, 4- and 5-transduced T cellsresponded in greater numbers to HCT-116 cells but also responded toNCI-H1975 cells.

EXAMPLE 7 Increased Cytotoxicity of MAGE-A3 Improved-AffinityTCR-Transduced T Cells in Response to a Tumour Cell Line than Wild Type

This assay is a colorimetric alternative to ⁵¹Cr release radioactivecytotoxicity assays and quantitatively measures lactate dehydrogenase(LDH) which is an enzyme that is released upon cell lysis. Released LDHin culture supernatants is measured with a 30-minute coupled enzymaticassay, which results in the conversion of a tetrazolium salt (INT) intoa red formazan product. The amount of colour formed is proportional tothe number of lysed cells. The absorbance data is collected using astandard 96-well plate reader at 490 nm.

Materials

-   -   CytoTox96® Non-Radioactive Cytotoxicity Assay (Promega) (G1780)        contains Substrate Mix, Assay Buffer, Lysis Solution, and Stop        Solution    -   Culture media: 10% FCS (heat-inactivated, Gibco, cat#10108-165),        88% RPMI 1640 with phenol red (Invitrogen, cat#42401042), 1%        glutamine, 200 mM (Invitrogen, cat#25030024), 1%        penicillin/streptomycin (Invitrogen cat#15070063)    -   Assay media: 10% FCS (heat-inactivated, Gibco, cat#10108-165),        88% RPMI 1640 without phenol red (Invitrogen, cat#32404014), 1%        glutamine, 200 mM (Invitrogen, cat#25030024), 1%        penicillin/streptomycin (Invitrogen cat#15070063)    -   Nunc microwell round bottom 96 well tissue culture plate (Nunc,        cat#163320)    -   Nunc-Immuno plates Maxisorb (Nunc, cat#442404)

Method

Target Cell Preparation

The targets cells (T) used in this assay were the HCT-116 colorectalcarcinoma cell line (HLA-A1⁺ MAGE-A3⁺) with or without knockdown ofMAGE-A3/6 protein expression by shRNA (knockdown performed as describedbelow). Target cells were prepared in assay medium: target cellconcentration was adjusted to 2×10⁵ cells/ml to give 1×10⁴ cells/well in50 μl.

MAGE-A3/6 Knockdown by siRNA

HCT-116 cells were transduced with lentiviral particles encodingMAGE-A3/6 shRNA (Santi Cruz Biotech, cat# sc-45284-V) as described inthe manufacturers' instructions. Briefly, 4×10⁴ cells were plated perwell of a 96 well flat bottom tissue culture plate (100 μl/well) andincubated overnight to adhere. Roughly 50% confluency was aimed for. Thefollowing day 10 μl medium was replaced with 10 μl of 100 μg/mlpolybrene (diluted in culture medium; Santa Cruz Biotech, cat#sc-134220) to give 5 μg/ml final concentration per well. The shRNAlentiviral particle-containing supernatant was defrosted slowly at roomtemperature and mixed gently. 60 μl of lentiviral particle-containingsupernatant was then added per well with a further 40 μl culture mediumto give 200 μl total volume per well, and cells incubated overnight.After 18 h medium was gently removed and replaced with 200 μl of freshculture medium without polybrene. The next day cells were detached with0.25% Trypsin-EDTA (Invitrogen, cat#25200) and seeded into 6 well platesfor expansion in fresh medium containing 5 μg/ml puromycin hydrochloride(Santa Cruz Biotech, cat# sc-108071) for selection of shRNA-expressingcells. Cells were frozen after several rounds of expansion. MAGE-A3/6protein expression knockdown was assessed by Western blot.

Effector Cell Preparation

The effector cells (E) used in this assay were mixed CD8⁺ and CD4⁺ Tcells (1:1) stimulated, transduced and expanded as described previously(Example 7). The effector to target ratio was 1.25:1. Effector cellswere prepared in assay medium; cell concentration was adjusted to2.5×10⁵/ml to give 1.25×10⁵ in 50 μl.

Assay Preparation

The constituents of the assay were added to the plate in the followingorder:

-   -   assay medium (to give 150 μl total per well)    -   50 μl of target cells (prepared as explained previously) to each        well    -   50 μl of effector cells (prepared as explained previously) to        each well

Several controls were prepared as explained below:

-   -   Effector spontaneous release: 50 μl effector cells alone.    -   Target cells spontaneous release: 50 μl target cells alone.    -   Target maximum release: 50 μl target cells alone+10 μl of        digitonin (600 μg/ml to give 40 μg/ml final)    -   Assay medium control: 150 μl medium alone.    -   Assay medium volume control for lysis solution: 150 μl medium+10        μl of digitonin.

All wells are prepared in triplicate in a final volume of 150 μl.

The plate was centrifuged at 250×g for 4 minutes then incubated at 37°C. for 24 hours. The plate was centrifuged at 250×g for 4 minutes. 50 μlof the supernatant from each well of the assay plate was transferred tothe corresponding well of a flat-bottom 96 well Nunc Maxisorb plate. TheSubstrate Mix was reconstituted using Assay Buffer (12 ml). 50 μl of thereconstituted Substrate Mix was then added to each well of the plate.The plate was covered with aluminium foil and incubated at roomtemperature for 30 minutes. 50 μl of Stop Solution was added to eachwell of the plate to stop the reaction. The absorbance at 490 nm wasrecorded on an ELISA plate reader within one hour after the addition ofStop Solution.

Calculation of Results

The average of absorbance values of the Culture Medium Background wassubtracted from all absorbance values of Experimental, Target CellSpontaneous Release and Effector Cell Spontaneous Release.

The average of the absorbance values of the Volume Correction Controlwas subtracted from the absorbance values obtained for the Target CellMaximum Release Control.

The corrected values obtained in the first two steps were used in thefollowing formula to compute percent cytotoxicity:

% cytotoxicity=100×(Experimental−Effector Spontaneous−TargetSpontaneous)/(Target Maximum−Target Spontaneous)

Results

The graph in FIG. 7 shows the specific killing of colorectal carcinomacells by T cells transduced to express TCR No:1, TCR No:2 or TCR No:3(as described in the table below). The TCR No:2-transduced T cells andTCR No:3-transduced T cells kill MAGE-A3-expressing HCT-116 colorectalcarcinoma cells, with an increased cytotoxicity compared to thewild-type TCR No:1-transduced T cells. This killing is reduced by shRNAknockdown of MAGE-A3/6 protein expression in the HCT-116 cells (T(M−)).

TCR α variable domain TCR β variable domain TCR No SEQ ID NO: SEQ ID NO:1 K1 to P114 of SEQ ID No: 2 K1 to T112 of SEQ ID No: 3 2 8 K1 to T112of SEQ ID No: 3 3 K1 to P114 of SEQ ID No: 2 10

1. (canceled)
 2. (canceled)
 3. (canceled)
 4. (canceled)
 5. (canceled) 6.A nucleic acid encoding a TCR having the property of binding to SEQ IDNO: 1-Human Leukocyte Antigen (HLA)-A1 complex and comprising a TCR αvariable domain and a TCR β variable domain, wherein: the TCR α variabledomain has the amino acid sequence from K1 to P114 of SEQ ID NO: 2 andthe TCR β variable domain has the amino acid sequence from K1 to T112 ofSEQ ID NO: 3 except that, in the TCR α variable domain, at least one ofthe following mutations is present, namely 50I is mutated to 50V; 51Q ismutated to 51R; 52S is mutated to 52P; 53S is mutated to 53Y; and/or inthe TCR β variable domain, at least one of the following mutations ispresent, namely 50F is mutated to 50T; 51S is mutated to 51D; 52E ismutated to 52M; 53T is mutated to 53L; 54Q is mutated to 54L.
 7. A cellcomprising a TCR expression vector comprising the nucleic acid asclaimed in claim 6 in a single open reading frame or two distinct openreading frames.
 8. A cell comprising a first expression vectorcomprising a nucleic acid encoding the alpha chain of the TCR of claim 6and a second expression vector comprising a nucleic acid encoding thebeta chain of the TCR of claim
 6. 9. A cell displaying on its surface aTCR encoded by the nucleic acid of claim
 6. 10. A cell as claimed inclaim 9 presenting a TCR having an α chain variable domain of SEQ ID NO:8 and a β chain variable domain of SEQ ID NO:
 3. 11. A cell as claimedin claim 9 presenting a TCR having an α chain variable domain of SEQ IDNO: 9 and a β chain variable domain of SEQ ID NO:
 3. 12. A cell asclaimed in claim 9 presenting a TCR having an α chain of SEQ ID NO: 2and a β chain variable domain of SEQ ID No:
 10. 13. A cell as claimed inclaim 9 presenting a TCR having an α chain of SEQ ID NO: 2 and a β chainvariable domain of SEQ ID NO:
 11. 14. A pharmaceutical compositioncomprising a plurality of cells as claimed in claim 7 together with oneor more pharmaceutically acceptable carriers or excipients.
 15. Anucleic acid as claimed in claim 6 wherein the TCR comprises one of theα chain variable domain amino acid sequences SEQ ID NOs: 8 and
 9. 16. Anucleic acid as claimed in claim 6 wherein the TCR comprises one of theβ chain variable domain amino acid sequences SEQ ID NOs: 10 and
 11. 17.A nucleic acid as claimed in claim 6 wherein the TCR also has an α chainTRAC constant domain and a β chain TRBC1 or TRBC2 constant domain, orhas an α chain TRAC and β chain TRBC1 or TRBC2 constant domains modifiedby truncation or substitution to delete the native disulfide bondbetween Cys4 of exon 2 of TRAC and Cyst of exon 2 of TRBC1 or TRBC2. 18.A nucleic acid as claimed in claim 6 wherein the TCR is an αβheterodimeric TCR, and has α and β chain constant domain sequences inwhich cysteine residues are substituted for Thr 48 of TRAC and Ser 57 ofTRBC1 or TRBC2, the said cysteines forming a disulfide bond between thealpha and beta constant domains of the TCR.